Nucleic acids encoding antagonistic CD40 monoclonal antibodies
The disclosure provides for antibodies that bind CD40, including a humanized antibody and a chimeric antibody with different Fc domains. The antibodies bind CD40 and do not exhibit CD40 agonist activity. The antibodies may comprise a modified IgG1 Fc domain, and exhibit minimal activation of immature dendritic cells. Compositions comprising antibodies, methods of use for treatment of diseases involving CD40 activity, and use in the preparation of a medicament for treatment of a disease involving CD40 activity are provided.
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This application is a divisional of U.S. patent application Ser. No. 15/988,554, filed May 24, 2018, now U.S. Pat. No. 11,220,550, issued Jan. 11, 2022, which claims the benefit of U.S. Provisional Application No. 62/511,079 filed May 25, 2017, each of which is hereby incorporated in its entirety for all purposes.
SEQUENCE LISTINGThe instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 22, 2021, is named 200896-0013-01-000030_SL.txt and is 186,769 bytes in size.
FIELDThe disclosure provides for antibodies that bind CD40, including a humanized antibody and a chimeric antibody with different Fc domains. The antibody polypeptides bind CD40 and do not exhibit CD40 agonist activity. The antibodies may comprise a modified IgG1 Fc domain, and exhibit minimal activation of immature dendritic cells. Compositions comprising antibodies, methods of use for treatment of diseases involving CD40 activity, and use in the preparation of a medicament for treatment of a disease involving CD40 activity are provided.
BACKGROUNDCD40 is a co-stimulatory molecule belonging to the tumor necrosis factor (TNF) receptor superfamily that is present on antigen presenting cells (APC), including dendritic cells, B cells, and macrophages. APCs are activated when CD40 binds its ligand, CD154 (CD40L), on TH cells. CD40-mediated APC activation is involved in a variety of immune responses, including cytokine production, up-regulation of co-stimulatory molecules (such as CD86), and enhanced antigen presentation and B cell proliferation. CD40 can also be expressed by endothelial cells, smooth muscle cells, fibroblasts, and epithelial cells.
CD40 activation is also involved in a variety of undesired T cell responses related to autoimmunity, transplant rejection, or allergic responses, for example. One strategy for controlling undesirable T cell responses is to target CD40 with an antagonistic antibody. For example, monoclonal antibody HCD122 (Lucatumumab), formerly known as Chiron 1212, is currently in clinical trials for the treatment of certain CD40-mediated inflammatory diseases. See “Study of HCD122 (Lucatumumab) and Bendamustine Combination Therapy in CD40+ Rituximab-Refractory Follicular Lymphoma,” Clinical Trials Feeds, on the Internet at hypertext transfer protocol: clinicaltrialsfeeds.org/clinical-trials/show/NCT01275209 (last updated Jan. 11, 2011). Monoclonal antibodies, however, can display agonist activity. For example, the usefulness of the anti-CD40 antibody, Chi220, is limited by its weak stimulatory potential. See Adams, et al., “Development of a chimeric anti-CD40 monoclonal antibody that synergizes with LEA29Y to prolong islet allograft survival,” J. Immunol. 174: 542-50 (2005).
SUMMARYIn a first embodiment, the present invention provides an isolated antibody, or antigen binding portion thereof, that specifically binds to human CD40, wherein the antibody comprises a heavy chain and a light chain, wherein said heavy chain comprises a CDR1 comprising GYTFTDLSMHW (SEQ ID NO: 1), a CDR2 comprising YITPSSGYTAYNQKFKG (SEQ ID NO: 2), a CDR3 comprising LILQRGAY (SEQ ID NO: 3); and said light chain comprises a CDR1 comprising RASKNVDSYGNSFMHW (SEQ ID NO: 4), a CDR2 comprising RASNLES (SEQ ID NO: 5), and a CDR3 comprising QQSNEDPLT (SEQ ID NO: 6).
In a second embodiment, the present invention provides an isolated antibody, or antigen binding portion thereof, that specifically binds to human CD40, wherein the antibody comprises a heavy chain and a light chain, wherein said heavy chain comprises a CDR1 comprising GYAFTNYLIE (SEQ ID NO: 17), a CDR2 comprising VINPGSGGTNYNEKFKG (SEQ ID NO: 18), and a CDR3 comprising SQLGRRFDY (SEQ ID NO: 19); and said light chain comprises a CDR1 comprising KASQDVRTGVA (SEQ ID NO: 20), a CDR2 comprising SASYRNT (SEQ ID NO: 21), and a CDR3 comprising QQHYSPPYT (SEQ ID NO: 22).
The isolated antibody or antigen binding portion thereof can antagonize activities of CD40. The isolated antibody or antigen binding portion thereof can be a chimeric antibody. The isolated antibody or antigen binding portion thereof can be a humanized antibody. The isolated antibody or antigen binding portion thereof can comprise a human heavy chain constant region and a human light chain constant region.
The isolated antibody or antigen binding portion thereof described herein can comprise a human IgG1 Fc domain comprising a mutation at Kabat position 238 that reduces binding to Fc-gamma-receptors (FcgRs), wherein proline 238 (P238) is mutated to one of the residues selected from the group consisting of lysine (K), serine (S), alanine (A), arginine (R) and tryptophan (W), and wherein the antibody or antigen binding portion thereof has reduced FcgR binding.
In certain embodiments, the isolated antibody or antigen binding portion thereof described herein can have P238 mutated to lysine (P238K) in a human IgG1 Fc domain. The isolated antibody or antigen binding portion thereof described herein, can comprise an Fc domain which comprises an amino acid sequence selected from:
In certain specific embodiments, the isolated antibody or antigen binding portion thereof described herein can comprise a human IgG1 Fc domain comprising an alanine substituted at Kabat position 297. The isolated antibody or antigen binding portion thereof described herein can comprise the Fc domain wherein the Fc domain comprises an amino acid sequence selected from:
In certain specific embodiments, the isolated antibody or antigen binding portion thereof comprises: (1) a variable heavy chain (VH) having the amino acid sequence of SEQ ID NO: 7 or (2) a variable light chain (VL) having the amino acid sequence of SEQ ID NO: 8. For example, the isolated antibody or antigen binding portion comprises: (1) a variable heavy chain (VH) having the amino acid sequence of SEQ ID NO: 7, and (2) a variable light chain (VL) having the amino acid sequence of SEQ ID NO: 8. In a specific embodiment, the isolated antibody or antigen binding portion thereof comprises (1) a heavy chain having the amino acid sequence of SEQ ID NO: 85, and (2) a light chain having the amino acid sequence of SEQ ID NO: 88. In other specific embodiments, the isolated antibody or antigen binding portion thereof comprises:
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- (1) a heavy chain having the amino acid sequence of SEQ ID NO: 106 and (2) a light chain having the amino acid sequence of SEQ ID NO: 88;
- (1) a heavy chain having the amino acid sequence of SEQ ID NO: 107 and (2) a light chain having the amino acid sequence of SEQ ID NO: 88;
- (1) a heavy chain having the amino acid sequence of SEQ ID NO: 108 and (2) a light chain having the amino acid sequence of SEQ ID NO: 88; or
- (1) a heavy chain having an amino acid sequence selected from SEQ ID NO: 84, 109, 110, or 111; and (2) a 5F11-45 light chain having the amino acid sequence of SEQ ID NO: 88.
In other specific embodiments, the isolated antibody or antigen binding portion thereof comprises (1) a variable heavy chain (VH) selected from the group consisting of: hz-HC1 (SEQ ID NO: 23), hz-HC2 (SEQ ID NO: 24), hz-HC3 (SEQ ID NO: 25), hz-HC4 (SEQ ID NO: 26), hz-HC5 (SEQ ID NO: 27), hz-HC6 (SEQ ID NO: 28), hz-HC7 (SEQ ID NO: 29), hz-HC8 (SEQ ID NO: 30), hz-HC9 (SEQ ID NO: 31), hz-HC10 (SEQ ID NO: 32), and hz-HC11 (SEQ ID NO: 33); and/or (2) a variable light chain (VL) selected from the group consisting of: hz-LC1 (SEQ ID NO: 34), hz-LC2 (SEQ ID NO: 35), hz-LC3 (SEQ ID NO: 36), hz-LC4 (SEQ ID NO: 37), and hz-LC5 (SEQ ID NO: 38).
In certain specific embodiments, the isolated antibody or antigen binding portion thereof comprises (1) a heavy chain amino acid sequence selected from SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 43, wherein the C-terminus optionally further comprises a lysine; and/or (2) a variable light chain (VL) amino acid sequence selected from SEQ ID NO: 41, and SEQ ID NO: 44. To illustrate, the isolated antibody or antigen binding portion thereof is selected from the group consisting of:
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- a) Y1238-hz1-P238K having a heavy chain of SEQ ID NO: 39 and light chain of SEQ ID NO: 41;
- b) Y1238-hz1-N297A having a heavy chain of SEQ ID NO: 40 and light chain of SEQ ID NO: 41;
- c) Y1238-hz2-P238K having a heavy chain of SEQ ID NO: 42 and light chain of SEQ ID NO: 44; and
- d) Y1238-hz2-N297A having a heavy chain of SEQ ID NO: 43 and light chain of SEQ ID NO: 44.
The antibody or antigen binding portion thereof disclosed herein, wherein the antigen binding portion is selected from the group consisting of Fv, Fab, F(ab′)2, Fab′, dsFv, scFv, sc(Fv)2, diabodies, and scFv-Fc.
The antibody or antigen binding portion thereof disclosed herein, wherein the antibody or antigen-binding portion thereof is linked to a therapeutic agent.
The antibody or antigen binding portion thereof disclosed herein, wherein the antibody or antigen-binding portion thereof is linked to a second functional moiety having a different binding specificity than said antibody or antigen binding portion thereof.
The antibody or antigen binding portion thereof disclosed herein further comprising an additional moiety.
A nucleic acid encoding an isolated antibody or antigen binding portion thereof is disclosed herein. An expression vector comprising the nucleic acid molecule is disclosed herein.
Also contemplated is a cell transformed with the expression vector.
Also disclosed is a method of preparing an anti-human CD40 antibody, or antigen binding portion thereof, comprising:
-
- a) expressing the antibody, or antigen binding portion thereof, in the cell transformed with the expression vector comprising the nucleic acid molecule encoding an isolated antibody or antigen binding portion thereof disclosed herein; and
- b) isolating the antibody, or antigen binding portion thereof, from the cell.
Also provided is a pharmaceutical composition comprising: a) the antibody, or antigen binding portion thereof disclosed herein; and b) a pharmaceutically acceptable carrier.
A method is provided of treating or preventing an immune response in a subject comprising administering to the subject the antibody, or the antigen binding portion thereof disclosed herein. In such method of treating or preventing an immune response in the subject, the subject has a disease selected from the group consisting of: Addison's disease, allergies, anaphylaxis, ankylosing spondylitis, asthma, atherosclerosis, atopic allergy, autoimmune diseases of the ear, autoimmune diseases of the eye, autoimmune hepatitis, autoimmune parotitis, bronchial asthma, coronary heart disease, Crohn's disease, diabetes, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, immune response to recombinant drug products (e.g., Factor VII in hemophiliacs), systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, transplant rejection, vasculitis, and ulcerative colitis.
Also contemplated is a use of an antibody, or antigen binding portion thereof as disclosed here, or a medicament comprising the same, for use to treat a subject in need thereof. Further contemplated is an antibody, or antigen binding portion thereof as disclosed herein in a therapeutically-effective amount, for use in treating or preventing an immune response, wherein the antibody or antigen binding portion thereof is for administering to a patient in need thereof.
The present disclosure is directed to anti-CD40 antibodies, and in particular, antagonistic anti-CD40 antibodies. For therapeutic targets such as CD40, FcgR-mediated cross-linking of anti-CD40 antibodies has the potential to lead to undesirable agonist signaling and potential for toxicity. The present disclosure also describes antagonistic anti-CD40 antibodies having reduced engagement of the “low affinity” FcgRs hCD32a/FcgRIIa, hCD32b/FcgRIIb, hCD16a/FcgRIIIa, and hCD16b/FcgRIIIb. Reduced engagement of low affinity FcgR's is expected to reduce the likelihood of undesirable agonist signaling and undesirable potential for toxicity.
Definitions & Abbreviations
-
- Further abbreviations and definitions are provided below.
- APC antigen presenting cells
- CD54 also referred to as ICAM-1
- CDR complementarity determining regions
- CH or CH constant heavy chain
- CL or CL constant light chain
- CHO cell Chinese hamster ovary cell
- dAb domain antibody
- FcgR interchangeable with FcγR
- FR Framework region
- GM-CSF granulocyte macrophage colony stimulating factor
- HC heavy chain
- iDC immature dendritic cells
- IFN interferon
- IgG Immunoglobulin G
- IL-6 Interleukin-6
- LC light chain
- mAb monoclonal antibody
- mg milligram
- ml or mL milliliter
- ng nanogram
- nM nanomolar
- pI isoelectric point
- SPR surface plasmon resonance
- TNF tumor necrosis factor
- μg microgram
- μM micromolar
- VL or VL variable light chain domain
- VH or VH variable heavy chain domain
Further abbreviations and definitions are provided herein.
In accordance with this detailed description, the following abbreviations and definitions apply. It must be noted that as used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an antibody” includes a plurality of such antibodies and reference to “the dosage” includes reference to one or more dosages and equivalents thereof known to those skilled in the art, and so forth.
As used here, the term “about” is understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. Generally, “about” encompasses a range of values that are plus/minus 10% of a referenced value unless indicated otherwise in the specification.
It is understood that any and all whole or partial integers between the ranges set forth are included herein.
CD40 is also known and referred to as B-cell surface antigen CD40, Bp50, CD40L receptor, CDw40, CDW40, MGC9013, p50, TNFRSF5, and Tumor necrosis factor receptor superfamily member 5. “Human CD40” refers to the CD40 comprising the following amino acid sequence:
As used herein, the term “variable domain” refers to immunoglobulin variable domains defined by Kabat et al., Sequences of Immunological Interest, 5th ed., U.S. Dept. Health & Human Services, Washington, D.C. (1991). The numbering and positioning of CDR amino acid residues within the variable domains is in accordance with the well-known Kabat numbering convention. VH, “variable heavy chain” and “variable heavy chain domain” refer to the variable domain of a heavy chain. VL, “variable light chain” and “variable light chain domain” refer to the variable domain of a light chain.
The term “human,” when applied to antibodies, means that the antibody has a sequence, e.g., FR and/or CH domains, derived from a human immunoglobulin. A sequence is “derived from” a human immunoglobulin coding sequence when the sequence is either: (a) isolated from a human individual or from a cell or cell line from a human individual; (b) isolated from a library of cloned human antibody gene sequences or of human antibody variable domain sequences; or (c) diversified by mutation and selection from one or more of the polypeptides above.
An “isolated” compound as used herein means that the compound is removed from at least one component with which the compound is naturally associated with in nature.
Antibodies of the present disclosure can be administered to human patients while largely avoiding the anti-antibody immune response often provoked by the administration of antibodies from other species, e.g., mouse. For example, murine antibodies can be “humanized” by grafting murine CDRs onto a human variable domain FR, according to procedures known in the art. Human antibodies as disclosed herein, however, can be produced without the need for genetic manipulation of a murine antibody sequence.
The anti-CD40 antibodies useful in the present disclosure comprise three complementarity-determining regions (CDRs) and four framework regions (FRs), arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs contain most of the residues that form specific interactions with the antigen and are primarily responsible for antigen recognition.
In a specific embodiment, the anti-CD40 antibodies of the present disclosure can comprise CDRs of humanized antibody 5F11-45. Amino acid sequences of 5F11-45 are provided in Table 1.
In one embodiment, the antibodies of the disclosure can comprise the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the humanized 5F11-45 variable heavy and light chains sequences (see e.g., SEQ ID NOS: 7 and 8 respectively, as an example). Monoclonal antibodies contain all 6 CDRs (3 for the VH and 3 for the VL), for example, GYTFTDLSMHW (SEQ ID NO: 1), YITPSSGYTAYNQKFKG (SEQ ID NO: 2), and LILQRGAY (SEQ ID NO: 3) for the variable heavy chain CDRs 1-3 respectively and RASKNVDSYGNSFMHW (SEQ ID NO: 4), RASNLES (SEQ ID NO: 5), and QQSNEDPLT (SEQ ID NO: 6) for the variable light chain CDRs 1-3 respectively.
In another embodiment, the antibodies of the disclosure can comprise the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the humanized Y1238 variable heavy and light chains sequences (see e.g., SEQ ID NOS: 23 and 34 respectively as examples). Monoclonal antibodies contain all 6 CDRs (3 for the VH and 3 for the VL), for example, GYAFTNYLIE (SEQ ID NO: 17), VINPGSGGTNYNEKFKG (SEQ ID NO: 18), and SQLGRRFDY (SEQ ID NO: 19) for the variable heavy chain CDRs 1-3 respectively and KASQDVRTGVA (SEQ ID NO: 20), SASYRNT (SEQ ID NO: 21), and QQHYSPPYT (SEQ ID NO: 22) for the variable light chain CDRs 1-3 respectively.
An “antibody” (Ab) shall include, without limitation, an immunoglobulin which binds specifically to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprises one constant domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
An “antigen binding portion” of an Ab (also called an “antigen-binding fragment”) or antigen binding portion thereof refers to one or more sequences of an Ab (full length or fragment of the full length antibody) that retain the ability to bind specifically to the antigen bound by the whole Ab. Examples of an antigen-binding fragment include Fab, F(ab′)2, scFv (single-chain variable fragment), Fab′, dsFv, sc(Fv)2, and scFv-Fc.
A “humanized” antibody refers to an Ab in which some, most or all of the amino acids outside the CDR domains of a non-human Ab are replaced with corresponding amino acids derived from human immunoglobulins. In one embodiment of a humanized form of an Ab, some, most or all of the amino acids outside the CDR domains have been replaced with amino acids from human immunoglobulins, whereas some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not abrogate the ability of the Ab to bind to a particular antigen. A “humanized” Ab retains an antigenic specificity similar to that of the original Ab.
A “chimeric antibody” refers to an Ab in which the variable regions are derived from one species and the constant regions are derived from another species, such as an Ab in which the variable regions are derived from a mouse Ab and the constant regions are derived from a human Ab.
As used herein, “specific binding” refers to the binding of an antigen by an antibody with a dissociation constant (Kd) of about 1 □M or lower as measured, for example, by surface plasmon resonance (SPR). Suitable assay systems include the BIAcore™ (GE Healthcare Life Sciences, Marlborough, Mass.) surface plasmon resonance system and BIAcore™ kinetic evaluation software (e.g., version 2.1).
Binding of the present antibodies to CD40 antagonizes CD40 activity. “CD40 activities” include, but are not limited to, T cell activation (e.g., induction of T cell proliferation or cytokine secretion), macrophage activation (e.g., the induction of reactive oxygen species and nitric oxide in the macrophage), and B cell activation (e.g., B cell proliferation, antibody isotype switching, or differentiation to plasma cells). CD40 activities can be mediated by interaction with other molecules. “CD40 activities” include the functional interaction between CD40 and the following molecules, which are identified by their Uniprot Accession Number is parentheses:
For example, a CD40 “activity” includes an interaction with TRAF2. CD40/TRAF2 interaction activates NF-κB and JNK. See Davies et al., Mol. Cell Biol. 25: 9806-19 (2005). This CD40 activity thus can be determined by CD40-dependent cellular NF-κB and JNK activation, relative to a reference.
As used herein, the terms “activate,” “activates,” and “activated” refer to an increase in a given measurable CD40 activity by at least 10% relative to a reference, for example, at least 10%, 25%, 50%, 75%, or even 100%, or more. A CD40 activity is “antagonized” if the CD40 activity is reduced by at least 10%, and in an exemplary embodiment, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, or even 100% (i.e., no detectable activity), relative to the absence of the antagonist. For example, an antibody may antagonize some or all CD40 activity, while not activating CD40. For example, the antibody may not activate B cell proliferation. The antibody may not activate cytokine secretion by T cells, where the cytokine is at least one cytokine selected from the group consisting of IL-2, IL-6, IL-10, IL-13, TNF-α, IFN-γ.
Variable domains may comprise one or more framework regions (FR) with the same amino acid sequence as a corresponding framework region encoded by a human germline antibody gene segment. Preferred framework sequences for use in the antibodies described herein are those that are structurally similar to the framework sequences used by antibodies described herein. The VH CDR1, 2 and 3 sequences, and the VL CDR1, 2 and 3 sequences, can be grafted onto framework regions that have the identical sequence as that found in the germline immunoglobulin gene from which the framework sequence derive, or the CDR sequences can be grafted onto framework regions that contain up to 20, preferably conservative, amino acid substitutions as compared to the germline sequences. For example, it has been found that in certain instances it is beneficial to mutate residues within the framework regions to maintain or enhance the antigen binding ability of the antibody (see e.g, U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).
Exemplary framework regions include but are not limited to those in TABLES 2 and 3 below. The sequences are in amino-to-carboxy terminus format. Exemplified heavy chain framework regions are shown in TABLE 2. A preferred group of heavy framework sequences for the humanized 5F11-45 VH is: QVQLVQSGAEVKKPGSSVKVSCKAS (FR1; SEQ ID NO: 46), WVRQAPGQGLEWMG (FR2; SEQ ID NO: 49), KTTLTADKSTSTAYMELSSLRSEDTAVYYCAR (FR3; SEQ ID NO: 51), and WGQGTLVTVSS (FR4: SEQ ID NO: 60). A preferred group of heavy framework sequences for the humanized Y1238 VH is: QVQLVQSGAEVKKPGASVKVSCKAS (FR1; SEQ ID NO: 47), WVRQAPGQGLEWMG (FR2; SEQ ID NO: 49), RVTMTRDTSISTAYMELSRLRSDDTAVYYCAR (FR3; SEQ ID NO: 52), and WGQGTLVTVSS (FW4; SEQ ID NO: 60).
Exemplified light chain framework regions are shown in TABLE 3. A preferred group of light chain framework regions for the humanized 5F11-45 VL include the following: DIVLTQSPDSLAVSLGERATINC (FR1; SEQ ID NO: 61), WYQQKPGQPPKLLIY (FR2; SEQ ID NO: 63), GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (FR3; SEQ ID NO: 66), and FGQGTKLEIK (FR4; SEQ ID NO: 71). A preferred group of light chain framework regions for the humanized Y1238 VL include the following: DIQMTQSPSSLSASVGDRVTITC (FR1; SEQ ID NO: 62), WYQQKPGKAPKLLIY (FR2; SEQ ID NO: 64), GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC (FR3; SEQ ID NO: 67), GVPSRFSGSRSGTDFTFTISSLQPEDIATYYC (FR3; SEQ ID NO: 68), and FGGGTKVEIK (FR4; SEQ ID NO: 72).
A variant variable domain may differ from the variable domain of the humanized 5F11-45 or Y1238 sequence by up to 10 amino acids or any integral value between, where the variant variable domain specifically binds CD40. Alternatively, the variant variable domain may have at least 90% sequence identity (e.g., at least 92%, 95%, 98%, or 99% sequence identity) relative to the sequence of the humanized 5F11-45 or Y1238 sequence, respectively. Non-identical amino acid residues or amino acids that differ between two sequences may represent amino acid substitutions, additions, or deletions. Residues that differ between two sequences appear as non-identical positions, when the two sequences are aligned by any appropriate amino acid sequence alignment algorithm, such as BLAST® (a registered trademark of the U.S. National Library of Medicine).
Exemplary CD40 antibodies of the present invention can include an isolated antibody, or antigen binding portion thereof, that specifically binds to human CD40, wherein said antibody comprises a heavy chain and a light chain, wherein:
-
- said heavy chain comprises a CDR1 comprising GYTFTDLSMHW (SEQ ID NO: 1), a CDR2 comprising YITPSSGYTAYNQKFKG (SEQ ID NO: 2), a CDR3 comprising LILQRGAY (SEQ ID NO: 3); and
- said light chain comprises a CDR1 comprising RASKNVDSYGNSFMHW (SEQ ID NO: 4), a CDR2 comprising RASNLES (SEQ ID NO: 5), and a CDR3 comprising QQSNEDPLT (SEQ ID NO: 6);
- or
- said heavy chain comprises a CDR1 comprising GYAFTNYLIE (SEQ ID NO: 17), a CDR2 comprising VINPGSGGTNYNEKFKG (SEQ ID NO: 18), and a CDR3 comprising SQLGRRFDY (SEQ ID NO: 19); and
- said light chain comprises a CDR1 comprising KASQDVRTGVA (SEQ ID NO: 20), a CDR2 comprising SASYRNT (SEQ ID NO: 21), and a CDR3 comprising QQHYSPPYT (SEQ ID NO: 22).
In one specific embodiment, an antibody or antigen binding portion thereof, that specifically binds to human CD40, comprises (1) a heavy chain comprising a CDR1 comprising GYTFTDLSMHW (SEQ ID NO: 1), a CDR2 comprising YITPSSGYTAYNQKFKG (SEQ ID NO: 2), a CDR3 comprising LILQRGAY (SEQ ID NO: 3); and/or (2) comprises a light chain comprising a CDR1 comprising RASKNVDSYGNSFMHW (SEQ ID NO: 4), a CDR2 comprising RASNLES (SEQ ID NO: 5), and a CDR3 comprising QQSNEDPLT (SEQ ID NO: 6). Optionally, such antibody or antigen binding portion thereof comprises (1) a variable heavy chain of SEQ ID NO: 7; and/or (2) a light variable chain of SEQ ID NO: 8. To illustrate, such antibody or antigen binding portion thereof comprises (1) a heavy chain comprising the variable heavy chain of SEQ ID NO: 7; and (2) a light chain comprising the variable light chain of SEQ ID NO: 8.
An antibody or antigen binding portion thereof, that specifically binds to human CD40 can comprise a heavy chain comprising the variable heavy chain of SEQ ID NO: 7, and a light chain comprising the variable light chain of SEQ ID NO: 8, wherein the heavy chain comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 98. An antibody or antigen binding portion thereof, that specifically binds to human CD40 can comprise a heavy chain comprising the variable heavy chain of SEQ ID NO: 7, and a light chain comprising the variable light chain of SEQ ID NO: 8, wherein the heavy chain comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 99.
An antibody or antigen binding portion thereof, that specifically binds to human CD40 can comprise (1) a heavy chain comprising the amino acid sequence of SEQ ID NO: 85, SEQ ID NO: 106, 107, or 108; and (2) a light chain comprising the amino acid sequence of SEQ ID NO: 88. To illustrate, such antibody or antigen binding portion thereof comprises (1) a heavy chain comprising the amino acid sequence of SEQ ID NO: 85; and (2) a light chain comprising the amino acid sequence of SEQ ID NO: 88.
The isolated antibody or antigen binding portion thereof can antagonize activities of CD40. The isolated antibody or antigen binding portion thereof can be a chimeric antibody. The isolated antibody or antigen binding portion thereof can be a humanized antibody. The isolated antibody or antigen binding portion thereof can comprise a human heavy chain constant region and a human light chain constant region.
The isolated antibody or antigen binding portion thereof described herein can comprise a human IgG1 Fc domain comprising a mutation at Kabat position 238 that reduces binding to Fc-gamma-receptors (FcgRs), wherein proline 238 (P238) is mutated to one of the residues selected from the group consisting of lysine (K), serine (S), alanine (A), arginine (R) and tryptophan (W), and wherein the antibody or antigen binding portion thereof has reduced FcgR binding. The isolated antibody or antigen binding portion thereof described herein can have P238 mutated to lysine in a human IgG1 Fc domain.
The isolated antibody or antigen binding portion thereof comprises an Fc domain which comprises an amino acid sequence selected from:
The isolated antibody or antigen binding portion thereof described herein can comprise a human IgG1 Fc domain comprising an alanine substituted at Kabat position 297. For example, the isolated antibody or antigen binding portion thereof comprises an Fc domain which comprises an amino acid sequence selected from:
The isolated antibody or antigen binding portion described herein, wherein the antibody comprises (1) a variable heavy chain (VH) selected from the group consisting of: hz-HCl (SEQ ID NO: 23), hz-HC2 (SEQ ID NO: 24), hz-HC3 (SEQ ID NO: 25), hz-HC4 (SEQ ID NO: 26), hz-HC5 (SEQ ID NO: 27), hz-HC6 (SEQ ID NO: 28), hz-HC7 (SEQ ID NO: 29), hz-HC8 (SEQ ID NO: 30), hz-HC9 (SEQ ID NO: 31), hz-HC10 (SEQ ID NO: 32), and hz-HC11 (SEQ ID NO: 33); and/or (2) a variable light chain (VL) selected from the group consisting of: hz-LC1 (SEQ ID NO: 34), hz-LC2 (SEQ ID NO: 35), hz-LC3 (SEQ ID NO: 36), hz-LC4 (SEQ ID NO: 37), and hz-LC5 (SEQ ID NO: 38).
The isolated antibody or antigen binding portion thereof disclosed herein, wherein the antibody comprises a heavy chain amino acid sequence selected from SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 43.
The isolated antibody or antigen binding portion thereof disclosed herein, wherein the antibody comprises a light chain amino acid sequence selected from SEQ ID NO: 41 and SEQ ID NO: 44.
The isolated antibody or antigen binding portion thereof disclosed herein, wherein the antibody is selected from the group consisting of:
-
- a) Y1238-hz1-P238K having a heavy chain of SEQ ID NO: 39 and light chain of SEQ ID NO: 41;
- b) Y1238-hz1-N297A having a heavy chain of SEQ ID NO: 40 and light chain of SEQ ID NO: 41;
- c) Y1238-hz2-P238K having a heavy chain of SEQ ID NO: 42 and light chain of SEQ ID NO: 44; and
- d) Y1238-hz2-N297A having a heavy chain of SEQ ID NO: 43 and light chain of SEQ ID NO: 44.
The antibody or antigen binding portion thereof disclosed herein, wherein the antigen binding portion is selected from the group consisting of Fv, Fab, F(ab′)2, Fab′, dsFv, scFv, sc(Fv)2, diabodies, and scFv-Fc.
The antibody or antigen binding portion thereof disclosed herein can be an immunoconjugate wherein the antibody or antigen-binding portion thereof is linked to a therapeutic agent.
The antibody or antigen binding portion thereof disclosed herein can be a bispecific antibody, wherein the antibody or antigen-binding portion thereof is linked to a second functional moiety having a different binding specificity than said antibody or antigen binding portion thereof.
The antibody or antigen binding portion thereof disclosed herein further comprising an additional moiety.
Fc Domain
The carboxy-terminal “half” of a heavy chain defines a constant region (Fc) and which is primarily responsible for effector function. As used herein, the term “Fc domain” refers to the constant region antibody sequences comprising CH2 and CH3 constant domains as delimited according to Kabat et al., Sequences of Immunological Interest, 5th ed., U.S. Dept. Health & Human Services, Washington, D.C. (1991). The Fc region may be derived from a human IgG. For instance, the Fc region may be derived from a human IgG1 or a human IgG4 Fc region. A heavy variable domain can be fused to an Fc domain. The carboxyl terminus of the variable domain may be linked or fused to the amino terminus of the Fc CH2 domain. Alternatively, the carboxyl terminus of the variable domain may be linked or fused to the amino terminus of a linker amino acid sequence, which itself is fused to the amino terminus of an Fc domain. Alternatively, the carboxyl terminus of the variable domain may be linked or fused to the amino terminus of a CH1 domain, which itself is fused to the Fc CH2 domain. Optionally, the protein may comprise the hinge region after the CH1 domain in whole or in part. Optionally an amino acid linker sequence is present between the variable domain and the Fc domain. The carboxyl terminus of the light variable domain may be linked or fused to the amino terminus of a CL domain.
An exemplary sequence for a heavy chain CH1 is amino acids 118-215 of SEQ ID NO: 85. An exemplary sequence for a light chain CL is amino acids 112-218 of SEQ ID NO: 88.
The antibody can be a fusion antibody comprising a first variable domain that specifically binds human CD40, and a second domain comprising an Fc domain.
Exemplary Fc domains used in the fusion protein can include human IgG domains. Exemplary human IgG Fc domains include IgG4 Fc domain and IgG1 Fc domain. While human IgG heavy chain genes encode a C-terminal lysine, the lysine is often absent from endogenous antibodies as a result of cleavage in blood circulation. Antibodies having IgG heavy chains including a C-terminal lysine, when expressed in mammalian cell cultures, may also have variable levels of C-terminal lysine present (Cai et al, 2011, Biotechnol Bioeng. 108(2):404-12). Accordingly, the C-terminal lysine of any IgG heavy chain Fc domain disclosed herein may be omitted.
The isolated antibody or antigen binding portion thereof described herein, can comprise an Fc domain which comprises an amino acid sequence of:
The Fc domain can comprise an amino acid sequence selected from the sequences in TABLE 4. SEQ ID NOS: 10, 12, 14, 16, 99, 101, 103 and 105 comprise a CH1 domain at the N-terminal (residues 1-98).
The variable regions of the present antibodies may optionally be linked to the Fc domain by an “amino acid linker” or “linker.” For example, the C-terminus of a variable heavy chain domain may be fused to the N-terminus of an amino acid linker, and an Fc domain may be fused to the C-terminus of the linker. Although amino acid linkers can be any length and consist of any combination of amino acids, the linker length may be relatively short (e.g., five or fewer amino acids) to reduce interactions between the linked domains. The amino acid composition of the linker also may be adjusted to reduce the number of amino acids with bulky side chains or amino acids likely to introduce secondary structure. Suitable amino acid linkers include, but are not limited to, those up to 3, 4, 5, 6, 7, 10, 15, 20, or 25 amino acids in length. Representative amino acid linker sequences include GGGGS (SEQ ID NO: 73), and a linker comprising 2, 3, 4, or 5 copies of GGGGS (SEQ ID NOs: 74-77, respectively). TABLE 5 lists suitable linker sequences for use in the present disclosure.
Antibody Preparation
The antibody can be produced and purified using ordinary skill in a suitable mammalian host cell line, such as CHO, 293, COS, NSO, and the like, followed by purification using one or a combination of methods, including protein A affinity chromatography, ion exchange, reverse phase techniques, or the like.
As well known in the art, multiple codons can encode the same amino acid. Nucleic acids encoding a protein sequence thus include nucleic acids having codon degeneracy. The polypeptide sequences disclosed herein can be encoded by a variety of nucleic acids. The genetic code is universal and well known. Nucleic acids encoding any polypeptide sequence disclosed herein can be readily conceived based on conventional knowledge in the art as well as optimized for production. While the possible number of nucleic acid sequence encoding a given polypeptide is large, given a standard table of the genetic code, and aided by a computer, the ordinarily skilled artisan can easily generate every possible combination of nucleic acid sequences that encode a given polypeptide.
A representative nucleic acid sequence coding a 5F11-45 heavy chain variable domain is:
In this sequence, nucleotides 76-105 encode CDR1, nucleotides 148-198 encode CDR2, and nucleotides 295-318 encode CDR3 of the 5F11-45 variable domain of the heavy chain.
A representative nucleic acid sequence coding a 5F11-45 light chain variable domain is:
In this sequence, nucleotides 70-117 encode CDR1, nucleotides 160-180 encode CDR2, and nucleotides 277-303 encode CDR3 of the 5F11-45 variable domain of the light chain.
A representative nucleic acid sequence coding a 5F11-45 heavy chain comprising a P238K Fc domain is:
caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaaggcttctggatacac cttcactgacttatcgatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggatacattactcctagcagtggatatactgc gtacaatcagaagttcaagggcaagaccacgttgaccgcggacaaatccacgagcacagcctacatggagctgagcagcctgagatctg aggacacggccgtgtattactgtgcgagattgatcttacaacggggagcttactggggccagggaaccctggtcaccgtctcctcagctag caccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggact acttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggccgtcctacagtcctcaggac tctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacac caaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaaagtc agtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaa gaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagca cgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcc cagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagct gaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccgg agaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggc agcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggt (SEQ ID NO: 115). In this sequence, nucleotides 1-351 encode the variable domain of the heavy chain. Nucleotides 352-645 encode the CH1 domain, and nucleotides 646-1338 encode the Fc domain. Optionally, a codon for lysine can be added to 3′ end of the Fc domain sequence.
A representative nucleic acid sequence coding a 5F11-45 light chain is:
In this sequence, nucleotides 1-333 encode the variable domain of the light chain, and nucleotides 334-654 encode CL.
The coding sequence for the heavy and/or light chain may encode a signal peptide, such as MRAWIFFLLCLAGRALA (SEQ ID NO: 119), at the 5′ end of the coding sequence. Exemplary coding sequences for a 5F11-45 heavy chain and light chain, each including a signal peptide coding sequence, are:
Accordingly, a nucleic acid encoding an antibody disclosed herein is also contemplated. Such a nucleic acid may be inserted into a vector, such as a suitable expression vector, e.g., pHEN-1 (Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137). Further provided is an isolated host cell comprising the vector and/or the nucleic acid.
The antibody of the disclosure can be produced and purified using only ordinary skill in any suitable mammalian host cell line, such as CHO (Chinese hamster ovary cells), 293 (human embryonic kidney 293 cells), COS cells, NSO cells, and the like, followed by purification using one or a combination of methods, including protein A affinity chromatography, ion exchange, reverse phase techniques, or the like.
Pharmaceutical Compositions and Methods of Treatment
A pharmaceutical composition comprises a therapeutically-effective amount of one or more antibodies and optionally a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers can further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers that enhance the shelf-life or effectiveness of the fusion protein. The compositions can be formulated to provide quick, sustained, or delayed release of the active ingredient(s) after administration. Suitable pharmaceutical compositions and processes for preparing them are known in the art. See, e.g., Remington, T
The pharmaceutical composition further may comprise an immuno-suppressive/immuno-modulatory and/or anti-inflammatory agent.
A method of treating an immune disease in a patient in need of such treatment may comprise administering to the patient a therapeutically effective amount of the antibody. Antagonizing CD40-mediated T cell activation could inhibit undesired T cell responses occurring during autoimmunity, transplant rejection, or allergic responses, for example. Inhibiting CD40-mediated T cell activation could moderate the progression and/or severity of these diseases.
Also provided is the use of an antibody of the disclosure, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treatment of an immune disease in a patient in need of such treatment. The medicament can, for example, be administered in combination with an immunosuppressive/immunomodulatory and/or anti-inflammatory agent.
As used herein, a “patient” means an animal, e.g., mammal, including a human. The patient may be diagnosed with an immune disease. “Treatment” or “treat” or “treating” refers to the process involving alleviating the progression or severity of a symptom, disorder, condition, or disease. An “immune disease” refers to any disease associated with the development of an immune reaction in an individual, including a cellular and/or a humoral immune reaction. Examples of immune diseases include, but are not limited to, inflammation, allergy, autoimmune disease, or graft-related disease. An “autoimmune disease” refers to any disease associated with the development of an autoimmune reaction in an individual, including a cellular and/or a humoral immune reaction. An example of an autoimmune disease is inflammatory bowel disease (IBD), including, but not limited to ulcerative colitis and Crohn's disease. Other autoimmune diseases include systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, diabetes, psoriasis, scleroderma, and atherosclerosis. Graft-related diseases include graft versus host disease (GVHD), acute transplantation rejection, and chronic transplantation rejection.
Diseases that can be treated by administering the antibody of the disclosure may be selected from the group consisting of Addison's disease, allergies, anaphylaxis, ankylosing spondylitis, asthma, atherosclerosis, atopic allergy, autoimmune diseases of the ear, autoimmune diseases of the eye, autoimmune hepatitis, autoimmune parotitis, bronchial asthma, coronary heart disease, Crohn's disease, diabetes, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, immune response to recombinant drug products (e.g., Factor VII in hemophiliacs), systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, transplant rejection, vasculitis, and ulcerative colitis.
The pharmaceutical composition may be administered alone or as a combination therapy, (i.e., simultaneously or sequentially) with an immunosuppressive/immunomodulatory and/or anti-inflammatory agent. Different immune diseases can require use of specific auxiliary compounds useful for treating immune diseases, which can be determined on a patient-to-patient basis. For example, the pharmaceutical composition may be administered in combination with one or more suitable adjuvants, e.g., cytokines (IL-10 and IL-13, for example) or other immune stimulators, e.g., chemokines, tumor-associated antigens, and peptides. Suitable adjuvants are known in the art.
Any suitable method or route can be used to administer the antibody or the pharmaceutical composition. Routes of administration include, for example, intravenous, intraperitoneal, subcutaneous, or intramuscular administration. A therapeutically effective dose of administered antibody depends on numerous factors, including, for example, the type and severity of the immune disease being treated, the use of combination therapy, the route of administration of the antibody or pharmaceutical composition, and the weight of the patient. A non-limiting range for a therapeutically effective amount of a domain antibody is 0.1-20 milligram/kilogram (mg/kg), and in an aspect, 1-10 mg/kg, relative to the body weight of the patient.
Kits
A kit useful for treating an immune disease in a human patient is provided. The kit can comprise (a) a dose of an antibody of the present disclosure and (b) instructional material for using the antibody in the method of treating an immune disease in a patient.
“Instructional material,” as that term is used herein, includes a publication, a recording, a diagram, or any other medium of expression, which can be used to communicate the usefulness of the composition and/or compound of the invention in a kit. The instructional material of the kit may, for example, be affixed to a container that contains the compound and/or composition of the invention or be shipped together with a container, which contains the compound and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively. Delivery of the instructional material may be, for example, by physical delivery of the publication or other medium of expression communicating the usefulness of the kit, or may alternatively be achieved by electronic transmission, for example by means of a computer, such as by electronic mail, or download from a website.
EXAMPLES Example 1: Fc-Formatting of Anti-CD40 Antibodies Impacts Activation of Immature Dendritic CellsAntibodies can be engineered to have different affinities and selectivity for FcgRs (FcγRs) by mutating the heavy chain constant region, such as in the hinge and Fc domain. Mutations can be introduced to either enhance or reduce FcgR binding. These mutations can increase or decrease FcgR-mediated cross-linking and/or signaling. For therapeutic targets such as CD40, FcgR-mediated cross-linking of anti-CD40 antibodies can has the potential to lead to undesirable agonist signaling and potential for toxicity. It is important to identify anti-CD40 antagonist antibodies that demonstrate reduced FcgR binding and potential for cross-linking and/or signaling, specifically, reduced engagement of the “low affinity” FcgRs hCD32a/FcgRIIa, hCD32b/FcgRIIb, hCD16a/FcgRIIIa, and hCD16b/FcgRIIIb. Engagement of the “high affinity” receptor CD64/FcgRI is generally believed to be of lower concern due to saturation of this receptor with serum IgG.
Antibody 5F11-45 is disclosed in U.S. Pat. Pub. 20160376371 (incorporated herein by reference) as a humanized antibody with high affinity to human CD40 that is engineered to have agonist activity, and the ability to bind preferentially to CD32a-R131 and CD32b. TABLE 6 below provides the amino acid sequences of the variable regions of the heavy and light chains of 5F11-45. In the heavy chain variable region, CDR1 is amino acids 26-35, CDR2 is amino acids 50-66, and CDR3 is amino acids 99-106. In the light chain variable region, CDR1 is amino acids 24-38, CDR2 is amino acids 54-60, and CDR3 is amino acids 93-101. The CDRs are underlined in each variable region.
To explore Fc formatting impact on activation of immature dendritic cells (iDC), the humanized anti-CD40 antibody 5F11-45 was formatted with a wild type human IgG1f isotype (5F11-45-IgG1f) as well as four isotypes with mutations designed to reduce FcgR binding (i.e., 5F11-45-IgG1.3f, 5F11-45-CTza, 5F11-45-P238K, 5F11-45-N297A). See SEQ ID NOS: 81-85 in TABLE 6. TABLE 6 further lists the heavy and light chain constant regions for each 5F11-45 antibody. The Fc domain is underlined in Table 6. The heavy chain variable region (amino acids 1-117) and the light chain variable region (amino acids 1-111) are in bolded font. The sequence (amino acids 118-215 of the heavy chain) between the variable region (in bolded font) and the Fc domain (underlined) comprises the CH1 domain in the heavy chain. The non-bolded sequence (amino acids 112-218) in the light chain comprises the CL domain.
As controls, the same 5F11-45 antibody was engineered for enhanced binding to human CD32a and CD32b proteins (5F11-45-SE; heavy (HC) and light chains (LC) are depicted as SEQ ID NOS: 86 and 88 respectively) or enhanced selectivity for hCD32b over hCD32a (5F11-45-V11; heavy and light chains are depicted SEQ ID NOS: 87 and 88 respectively).
All 5G11-45 Fc variants demonstrated high affinity binding to CD40 target by surface plasmon resonance (SPR) TABLE 7.
The FcgR binding profiles of the Fc-engineered 5F11-45 antibodies were characterized by SPR to determine if the Fc mutations had the desired impacts on FcgR binding and selectivity. The SPR data for FcgR binding are shown in TABLE 8.
As shown in TABLE 8, 5F11-45-IgG1f binds to all FcgRs, with particularly strong binding to CD64, CD16a-V158, and CD32a-H131. The S267E variant (5F11-45-SE) (control) demonstrates the expected enhanced binding to CD32a and CD32b proteins. The five mutations present in the IgG1 V11 isotype of 5F11-45-V11 provided enhanced selectivity for CD32b.
The other four 5F11-45 Fc variants shown reduced FcgR binding. 5F11-45-IgG1.3f demonstrated reduced binding to all FcgRs including the weakest binding to CD64. At very high antibody concentrations (i.e., 10 μM), some weak binding of 5F11-45-IgG1.3f to CD32a-R131 is observed. In contrast, the other three variants (5F11-45-CTza, 5F11-45-P238K, and 5F11-45-N297A) demonstrated nearly undetectable binding to any of the low affinity FcgRs, even at a 10 μM antibody concentration. See TABLE 7 above. Differences were also observed in CD64 binding, especially in the dissociation rates, which were considerably faster for 5F11-45-P238K and 5F11-45-N297A compared to 5F11-45-CTza or 5F11-45-IgG1f,
Immature dendritic cells (iDC) are sensitive to indirect activation of CD40 through clustering/cross-linking which can lead to activation of immature DCs leading to cytokine release (e.g., interleukin-6, 11-6) and cell surface activation signal upregulation (e.g., CD86 and CD54). Chinese hamster ovary (CHO) cells highly over-expressing CD32a, and having a low affinity to FcgR were used to demonstrate the potential for FcgR-mediated clustering/cross-linking. The ratio of CHO cells to iDCs is 1:6, representing an exaggerated level of clustering/cross-linking.
BMS-986090 is an anti-CD40 antagonist domain antibody fused to IgG4 Fc domain and has the amino acid sequence:
(which is SEQ ID NO: 1287 in WO 2012/145673). The solid underlined portion of the BMS-986090 sequence corresponds to BMS3h-56-269 domain antibody (which is SEQ ID NO: 417 in WO 2012/145673). The dotted underline “AST” (SEQ ID NO: 78) is a linker and the rest is the Fc domain called IgG4.1. 2141 (mAb 134-2141) is a CD40 agonist antibody (see Robert Vonderheide et al., “Clinical Activity and Immune Modulation in Cancer Patients Treated with CP-870,893, a Novel CD40 Agonist Monoclonal Antibody,” 25(7): 876-883 (2007)). BMS-986090 and 2141 were used as positive controls for cross-linking mediated anti-CD40 iDC activation. L6-IgG4, a fusion protein with no CD40 binding capability, served as a negative control. As seen in
To further assess these observations in other antibodies, a potent mouse anti-human-CD40 antibody Y1238 was formatted as a chimeric antibody with these isotypes and as IgG1.3f (SEQ ID NOS: 90-93 for the heavy chains and SEQ ID NO: 94 for the light chains). See TABLE 9. The heavy chain variable region is amino acids 1-118 (bolded font). In the heavy chain variable region, CDR1 is amino acids 26-36, CDR2 is amino acids 50-66, and CDR3 is amino acids 99-107. The Fc domain is amino acids 217-448. The sequence (amino acids 119-216 of the heavy chain) between the variable region (in bolded font) and the Fc domain (underlined) comprises the CH1 domain in the heavy chain. The light chain variable region is amino acids 1-107. In the light chain variable region, CDR1 is amino acids 24-34, CDR2 is amino acids 50-56, and CDR3 is amino acids 89-97. Amino acids 108-214 in the light chain comprise the CL domain.
These chimeric antibodies were tested for CD40 and FcgR binding by SPR, as well as for iDC activation. All of the Fc variant Y1238 chimeric antibodies demonstrated high affinity for human-CD40, TABLE 10.
Like 5F11-45, the chimeric Y1238 antibodies with engineered Fc domains demonstrated much weaker FcgR binding as compared to the IgG1f isotype control. The Y1238-IgG1.3f molecule demonstrated some weak but measurable binding responses towards the low affinity FcgRs, in particular CD32a-R131 and CD32b. In contrast, the CTza, P238K and N297A variants had very weak or undetectable binding to these FcgRs, even at 10 μM antibody concentration, TABLE 11.
CD64 binding was similar for the chimeric Y1238 antibodies as with the 5F11-45 variants, with IgG1.3f demonstrating the weakest CD64 binding. Both the P238K and N297A variants have significantly faster dissociation rates than CTza, whether in the Y1238 chimeric antibody form or in the 5F11-45.
Like 5F11-45, the chimeric Y1238 demonstrated no activation of iDC either alone or with the addition of FcgR (CD32) over expressing CHO cells when formatted with the CTza, P238K, or N297A isotypes,
Chimeric Y1238-P238K and Y1238-N297A were further tested in iDC from additional donors, confirming that these molecules do not activate immature dendritic cells either alone or in the addition of CD32 CHO cells,
Humanization background/procedure is as discussed in section “II. Engineered and Modified Antibodies” in WO2017004006, which is incorporated herein by reference in its entirety. Based on this analysis, 11 VH sequences (SEQ ID NOS: 23-33) and 5 Vκ sequences (SEQ ID NOS: 34-38) were selected for testing. Substitutions are indicated in bold and by dotted underline. The CDRs in each variable heavy and variable light, in CDR1-CDR2-CDR3 orientation, are indicated by underlining, i.e., GYAFTNYLIE (SEQ ID NO: 17), VINPGSGGTNYNEKFKG (SEQ ID NO: 18), and SQLGRRFDY (SEQ ID NO: 19) respectively for the variable heavy CDRs (Table 12) and KASQDVRTGVA (SEQ ID NO: 20), SASYRNT (SEQ ID NO: 21), and QQHYSPPYT (SEQ ID NO: 22) for the variable light CDRs respectively (Table 13).
The VH sequences were formatted in the context of an IgG1.3f isotype. The Vκ sequences were formatted as a full light chain with a common CL sequence. All 55 combinations of these humanized HC and LC constructs, as well as the chimeric Y1238 molecule were expressed as 3 ml supernatants for binding analysis by SPR. Each of the 56 supernatants, as well as a purified Y1238-chimeric IgG1 antibody, was captured on a protein A sensor chip and tested for binding to two concentrations (i.e., 200 nM and 1000 nM) of hCD40-monomer using BIAcore™ SPR in a screening assay. All antibodies, except for those containing hz-HC3, captured at high levels (>300 RU) using a 100-fold dilution of the expression supernatant, indicating a high antibody titer in the supernatant except for the hz-HC3 samples. The kinetic data for the two concentrations of hCD40-monomer were fit to a 1:1 Langmuir model, to yield estimates of the kinetic and affinity values for these interactions, and for comparison of the different molecules. These data showed that the captured antibodies bound with high affinity to CD40; the estimated KD values between 2.65 nM and 18.2 nM, see TABLE 14. For a given light chain, the antibodies with heavy chains hzHC1, hz-HC2, hz-HC4, hz-HC5, hz-HC6, hz-HC7, hz-HC8 and hz-HC9 demonstrated higher CD40 affinity than those containing hz-HC10 or hz-HC11. Also, for a given heavy chain, the antibodies with light chains hz-LC1 or hz-LC2 had higher CD40 affinity than those with hz-LC3, hz-LC4 or hz-LC5. A full kinetic titration series of CD40 binding to representative supernatants and purified chimeric Y1238 also demonstrated that the estimated KD values based on the 2 point (200 nM and 1000 nM) screening data, were reasonable estimates of the binding kinetics and affinity for the supernatant samples when compared to a full kinetic titration series of analyte as shown in TABLE 14.
Based on the supernatant screening SPR data and bias towards constructs with fewer framework back-mutations, a subset of humanized Y1238 antibodies were selected for expression, purification, and further characterization. Two of these antibodies (i.e., Y1238-hz1 and Y1238-hz2) were formatted with P238K and N297A isotypes (i.e., SEQ ID NOS: 39, 40, 42, and 43 for heavy chains; see TABLE 15 for light chains) and tested for CD40 and FcgR binding by SPR. The CD40 binding SPR data showed that each antibody bound the CD40 target with high affinity, TABLE 16. The data is compared to that of another anti-CD40 antibody, antibody BI-mAb-B (i.e., SEQ ID NOS: 95 and 96, disclosed in U.S. Pat. No. 9,090,696 and referred to therein as “Antibody B”), which binds with much lower affinity than the humanized Y1238 molecules, TABLE 15. The Fc domain is underlined.
The alignment of the two light chain sequences for Hz1 and Hz2 from TABLE 15 (SEQ ID NOS: 41 and 44) is as follows and displays a one mutation difference (at position 66; underlined below) between the two sequences:
All the Y1238 antibodies are demonstrated potent antagonists of B cell proliferation driven by either soluble CD40L trimer or cellular CD40L from CD40L-expressing CHO cells (see TABLE 17). In contrast, the competitor antibody BI-mAb-B and 5F11-45-P238K showed potent inhibition of B cell proliferation driven by soluble CD40L signals, but was less effective at inhibiting B cell proliferation driven by cellular CD40L (CHO cells overexpressing CD40L). The Y1238 chimeric and humanized antibodies exhibited only about a 10-fold shift in potency when cellular CD40L was used to stimulated B cells.
FcgR binding for the humanized Y1238 antibodies (i.e., Y1238-hz1-N297A, Y1238-hz1-P238K, Y1238-hz2-P238K and Y1238-hz1-N297A) were consistent with previous data for the chimeric Y1238 antibodies and 5F11-45 antibodies, showing very weak binding responses for all of the low affinity FcgRs, TABLE 18.
In addition, humanized versions of Y1238 antibodies exhibited minimal activation of iDC over the negative control protein L6-IgG4 when tested either alone or with the addition of FcgR CD32 expressing CHO cells. This observation is consistent with the inability of the humanized Y1238 antibodies to activate CD40 even with the exaggerated cross-linking provided by the CD32 overexpressing CHO cells. In contrast, both the 2141 and BMS-986090 controls showed donor-dependent moderate activation of iDC alone which was robustly increased when cross-linked or clustered by the CD32 CHO cells,
FCGR BINDING SPR: FcgR binding can be measured in vitro using purified FcgRs using methods such as BIAcore™ surface plasmon resonance (SPR). One method tests the binding of purified antibodies to His-tagged FcgR proteins (FcgR-His) which are captured on the immobilized Fab fragment of an anti-His antibody. These experiments are performed on either a BIAcore™ T100 or BIAcore™ T200 instrument (GE Healthcare Life Sciences, Marlborough, Mass.) at 25° C. The Fab fragment from a murine anti-6×His antibody (generated in house) is immobilized on a CMS sensor chip using standard ethyl(dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) chemistry with ethanolamine blocking, to a density of ˜3000 RU in a running buffer of 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant p20 (HBS-EP+). All remaining studies are performed using a running buffer of 10 mM NaPO4, 130 mM NaCl, 0.05% p20 (PBS-T) pH 7.1. Various FcγR proteins containing a C-terminal 6× poly-histidine tag (generated in house) are captured on this surface (typically using FcgR-his protein concentration of ˜7 μg/ml) using a contact time of 30 sec at 10 μl/min. Various concentrations of purified antibody are tested for binding, for example using an association time of 120 seconds at 30 μl/min, and a dissociation time of 120 seconds at 30 μl/min. FcgR proteins tested in these studies include the “high affinity” FcgR CD64 (hFcgRI), as well as the “low affinity” FcgRs CD32a-H131 (FcgRIIa-H131), CD32a-R131 (FcgRIIa-R131), CD32b (FcgRIIb), CD16a-V158 (FcgRIIIa-V158), and CD16b-NA2 (FcgRIIIb-NA2).
To quantitatively analyze the binding responses and compare the FcgR binding of different molecules, the SPR binding data can be analyzed by calculating the maximum binding response as a percentage of the theoretical maximum binding response (% Rmax), using the equation:
% R max=(Binding Response Analyte)/[((Mw Analyte)/(Mw Ligand))×(Response Ligand)×(analyte:ligand stoichiometry)] EQUATION1:
where “Analyte” is the antibody and Ligand is the captured FcgR protein.
This analysis does not take into account the mass of glycosylation of antibody or FcgR, and assumes 100% fractional activity for the captured ligand. Since the FcgRs are glycosylated, the % Rmax values are typically less than 100% even under saturating conditions. The % Rmax analysis is particularly useful for evaluating the binding of the “low affinity” FcgRs CD32a-H131, CD32a-R131, CD32b, CD16a-V158 and CD16b-NA2, which have relatively fast association and dissociation rates and affinities near or below the analyte concentrations tested (1 uM), so saturation of the surface is generally not achieved under these conditions. In contrast, the “high affinity” FcgR CD64 binds with higher affinity and slower dissociation kinetics than the other FcgRs particularly to IgG1 and IgG4. Thus, these isotypes do typically saturate the CD64 surface under micromolar analyte concentrations, and are more difficult to differentiate binding affinities using % Rmax. For these interactions, differences between antibodies can be easily observed by comparison of the dissociation rates in the sensorgram data.
CD40 BINDING KINETICS AND AFFINITY: The monovalent CD40 binding affinity of the antibody molecules is measured by SPR on a BIAcore™ T100 or T200 instrument (GE Healthcare) at 25° C. by capturing antibody on an immobilized protein A sensor chip surface, and then binding human-CD40-monomer protein (generated in house) using an association time of 180 seconds, dissociation time of 360 seconds at 30 μl/min in PBS-T (phosphate buffered saline with Tween®20) at pH 7.1.
PRIMARY CELL ISOLATION AND CULTURE: PBMCs (peripheral blood mononuclear cells) are isolated from heparinized human blood by Ficoll density gradient separation. Monocytes are isolated from PBMC following the Manual EasySep protocol (STEMCELL, Vancouver, Canada). One million of isolated monocytes are plated in in each well of a 6-well plate in 6 mls of complete media (RPMI-1640, 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin-streptomycin), containing human IL-4 (Interleukin-4, 100 ng/ml) and human GM-CSF (Granulocyte macrophage colony-stimulating factor, 100 ng/ml) and incubated for 6 days at 37° C./5% CO2, changing media every other day and replacing it with fresh media containing the same concentration of cytokines. iDCs were harvested on day 6, washed thoroughly, and re-suspended in complete media.
TREATMENT OF iDCS WITH ANTI-CD40 ANTIBODIES IN THE PRESENCE OR ABSENCE OF FcγR CLUSTERING/CROSSLINKING: Titrations of the various biological agents are made in complete media, and added to duplicate 96-well plates. In the case of cross-linking, antibodies are added to the cells for 30 min. prior to the addition of CD32a-expressing CHO cells at a ratio of 1:6. Cells were incubated at 37° C. at 5% CO2 for approximately 18-20 hours. 150 μL of supernatant is removed from each well, diluted 1:5, and is evaluated for protein concentrations of IL-6, TNFα and IL-12 using a commercially available ELISA kits (R&D Systems, Minneapolis, Minn.), according to manufacturer's instructions. The cells remaining in the plates from the harvested supernatants, are combined into 1 sample per duplicate treatment, and transferred to a new 96-well round bottom (RB) plate, and placed at 4° C. Cells are washed with D-PBS (Dulbecco's Phosphate Buffered Saline), Ca++ and Mg++ free, and stained for 30 min. on ice for cell viability using the LIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit (Invitrogen, Carlsbad, Calif.). Cells are washed and re-suspended in D-PBS, Ca++ and Mg++ free, 2% fetal bovine serum (FBS), 0.1% NaN3 (staining buffer) and blocked with 5 μl/well of Human TruStain FcX™ (Fc Receptor Blocking Solution, Biolegend, San Diego, Calif.) in staining buffer.
DCs are immuno-stained with: PerCpCy5.5-conjugated αCD3, αCD19, αCD14 (Lin−), BUV395-conjugated αCD11c (BD Biosciences, San Diego, Calif.), APC-conjugated αCD86 (Biolegend, San Diego, Calif.), PE-conjugated αCD83 (eBioscience, San Diego, Calif.), FITC-conjugated αCD54 (Biolegend, San Diego, Calif.), and incubated at 4° C. for 45 minutes. Cells are washed twice in staining buffer and fixed (15 at RT, protected from light), by adding 100 μl of BD Cytofix Fixation Buffer (BD Bioscience, San Diego, Calif.). DCs are evaluated for CD86, ICAM-1 and CD83 expression using a LSRII-Fortessa Flow Cytometer (BD Biosciences, San Diego, Calif.), and FlowJo analysis software (Treestar, Ashland, Oreg.).
Inhibition of CD40L induces human B cell proliferation: Human tonsillar B cells are obtained from pediatric patients during routine tonsillectomy. The cells are isolated by mincing and gently mashing the tissue, passing the cells through a screen and isolating mononuclear cells with density gradient separation using human Lympholyte®-H separation media (Cedarlane Labs, Burlington, ON). Mononuclear cells are collected from the interface, washed, and rosetted with sheep red blood cells (SRBC, Colorado Serum Company; Denver, Colo.) for one hour at 4° C., followed by density gradient separation to remove T cells. Cells are again washed and re-suspended in RPMI containing 10% FBS (complete media). Titrations of antibodies are made in complete media, and added in triplicate to 96-well round bottom (RB) plates. 1×105 tonsillar human B cells are added and stimulated with either soluble IZ-hCD40L (2 μg/mL), or with Chinese hamster ovary cells stably transfected with human CD40L (CHO-hCD40L) irradiated with 10,000 Rads; cells are then plated at 2×103 cells/well, in a final volume of 200 μL in each well. Plates are incubated at 37° C. at 5% CO2 for 72 hours, labeled for the last 6 hours with 0.5 μCi of 3[H]-thymidine per well, harvested, and counted by liquid scintillation. B cell proliferation was quantitated based on thymidine incorporation.
Claims
1. A nucleic acid encoding an isolated antibody or antigen binding portion thereof, that specifically binds to human CD40, wherein said antibody comprises a heavy chain, and a light chain, wherein:
- said heavy chain comprises a CDR1 comprising GYTFTDLSMHW (SEQ ID NO: 1), a CDR2 comprising YITPSSGYTAYNQKFKG (SEQ ID NO: 2), a CDR3 comprising LILQRGAY (SEQ ID NO: 3) and a human heavy chain constant region; and
- said light chain comprises a CDR1 comprising RASKNVDSYGNSFMHW (SEQ ID NO: 4), a CDR2 comprising RASNLES (SEQ ID NO: 5), and a CDR3 comprising QQSNEDPLT (SEQ ID NO: 6) and a human light chain constant region, and
- said human heavy chain constant region is a human IgG1 Fc domain comprising either (1) a mutation at Kabat position 238 that reduces binding to Fc-gamma-receptors (FcgRs), wherein proline 238 (P238) is mutated to one of the residues selected from the group consisting of lysine, serine, alanine, arginine and tryptophan, and wherein the antibody or antigen binding portion has reduced FcgR binding; or (2) or an alanine substituted at Kabat position 297.
2. An expression vector comprising the nucleic acid molecule of claim 1.
3. A cell transformed with the expression vector of claim 2.
4. A method of preparing an anti-human CD40 antibody, or antigen binding portion thereof, comprising:
- a) expressing the antibody, or antigen binding portion thereof, in the cell of claim 3; and
- b) isolating the antibody, or antigen binding portion thereof, from the cell.
5. The nucleic acid of claim 1, wherein the antibody or antigen binding portion thereof antagonizes activities of CD40.
6. The nucleic acid of claim 1, wherein said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8.
7. The nucleic acid of claim 1, comprising a human IgG1 Fc domain comprising a mutation at Kabat position 238 that reduces binding to Fc-gamma-receptors (FcgRs), wherein proline 238 (P238) is mutated to one of the residues selected from the group consisting of lysine, serine, alanine, arginine and tryptophan, and wherein the antibody or antigen binding portion has reduced FcgR binding.
8. The nucleic acid of claim 1, wherein P238 is mutated to lysine.
9. The nucleic acid of claim 1, wherein the Fc domain comprises an amino acid sequence selected from: SEQ ID NO: 98 (IgG1a-P238K (-C-term Lys)), SEQ ID NO: 9 (IgG1a-P238K), SEQ ID NO: 99 (CH1-IgG1a-P238K (-C-term Lys)), SEQ ID NO: 10 (CH1-IgG1a-P238K), SEQ ID NO: 100 (IgG1f-P238K (-C-term Lys)), SEQ ID NO: 11 (IgGlf-P238K), SEQ ID NO: 101 (CH1-IgG1f-P238K (-C-term Lys)), or SEQ ID No: 12 (CH1-IgGlf-P238K).
10. The nucleic acid of claim 1, wherein P238 is mutated to serine.
11. The nucleic of claim 6, wherein the human IgG1Fc domain comprises the amino acid sequence of SEQ ID NO: 98 or SEQ ID NO: 100.
12. The nucleic acid of claim 1, wherein the heavy chain comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 85, SEQ ID NO: 106, SEQ ID NO: 107, and SEQ ID NO: 108; and the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 88.
13. The nucleic acid of claim 1, wherein the isolated antibody or antigen binding portion thereof is humanized.
14. The nucleic acid of claim 1, wherein the antibody or antigen-binding portion thereof is linked to a therapeutic agent.
15. The nucleic acid of claim 1, wherein the antibody or antigen-binding portion thereof is linked to a second functional moiety having a different binding specificity than said antibody or antigen binding portion thereof.
16. The nucleic acid of claim 1, comprising a human IgG1 Fc domain comprising an alanine substituted at Kabat position 297.
17. The nucleic acid of claim 16, wherein the Fc domain comprises an amino acid sequence selected from: SEQ ID NO: 102 (IgG1a-N297A (-C-term Lys)), SEQ ID NO: 13 (IgG1a-N297A), SEQ ID NO: 103 (CH1-IgG1a-N297A (-C-term Lys)), SEQ ID NO: 14 (CH1-IgG1a-N297A), SEQ ID NO: 104 (IgG1f-N297A (-C-term Lys)), SEQ ID NO: 15 (IgGlf-N297A), SEQ ID NO: 105 (CH1-IgG1f-N297A (-C-term Lys)), or SEQ ID NO: 16 (CH1-IgGlf-N297A).
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Type: Grant
Filed: Dec 23, 2021
Date of Patent: Mar 28, 2023
Patent Publication Number: 20220177596
Assignee: Bristol-Myers Squibb Company (Princeton, NJ)
Inventors: Aaron Yamniuk (Vancouver), Mary Struthers (Edison, NJ), Suzanne J. Suchard (Portland, OR)
Primary Examiner: Phillip Gambel
Application Number: 17/561,529
International Classification: C07K 16/28 (20060101); A61K 39/395 (20060101);
